The optical microscope remains an irreplaceable tool for life science research even today, because light is often the best way to examine living cells non-invasively. As a special optical imaging technique, dark field (DF) microscopy enables the creation of high contrast images of unstained transparent specimens. In order to form a bright specimen image on a dark background, oblique rays from every azimuth are allowed to strike the samples but only light scattered from the specimens are collected by the objective. In other words, dark field (DF) microscopy works on the principle of illuminating a sample with light that will not be collected by the objective lens, and that will therefore not form part of the image. Consequently, the dark field image appears as a dark, almost black background with a bright object on it. Conventional approaches to DF microscopy typically involve an optical condenser capable of focusing light at a specific angle to the sample, and an objective that excludes the directional transmission of light through the sample.
Wide field fluorescence microscopy includes techniques that conventionally rely on illumination of fluorophore-labeled specimens with a broad cone of light. The limited spatial resolution demonstrated by wide field fluorescence microscopy, especially along the optical axis (referred to as “Z-resolution”), can create challenges in differentiating between individual specimen details that are overpowered by background fluorescence outside of the focal plane.